To study the mechanism of secretion and its regulation by Ca2+, we have been using digitonin-permeabilized PC12 cells and bovine chromaffin cells. The release of catecholamines from these permeabilized cells is both Ca2+- and ATP-dependent. We have previously proposed that norepinephrine secretion by PC12 cells is regulated by a Ca2+-stimulated phosphorylation. We have tested this model by adding protein phosphatases and phosphatase inhibitors to permeabilized PC12 cells. The effects of added phosphatases and phosphatase inhibitors on norepinephrine secretion are consistent with secretion in permeabilized PC12 cells being regulated by a Ca2+-stimulated phosphorylation. To identify proteins which are involved in the regulation of secretion we have been using digitonin-permeabilized bovine chromaffin cells. Prolonged incubation of permeabilized bovine chromaffin cells results in the loss of Ca2+-dependent norepinephrine secretion. We have found that the addition of a cytosolic extract to these depleted cells restores Ca2+-dependent secretion. We are currently trying to isolate the proteins in this extract which are responsible for resorting secretory activity. As reported last year, we find that GTP S induces Ca2+-independent norepinephrine secretion in permeabilized PC12 cells. The GTP-binding protein responsible for this stimulation does not appear to be one of the well characterized heterotrimeric G-proteins. The addition of a crude mixture of GTP-binding proteins to permeabilized PC12 cells results in an enhancement of GTP S-stimulated norepinephrine secretion. We are currently characterizing this enhancement and starting to fractionate GTP-binding proteins to determine which is responsible for this stimulation.